The present invention relates to the expression of cytochrome P450 in bacteria; in particular the invention relates to the expression of an active eukaryotic cytochrome P450 enzyme system in bacteria, particularly Enterobacteria.
Cytochrome P450 monooxygenases (P450s) form a superfamily of haemoproteins which catalyse the metabolism of a wide range of compounds. They catalyse the oxidation of lipophilic chemicals through the insertion of one atom of molecular oxygen into the substrate (Porter and Coon (1991) J. Biol. Chem. 261, 13469-13472). Mammalian P450s catalyse the metabolism of endogenous and exogenous compounds, including steroids, therapeutic drugs and carcinogens (Guengerich (1987) in xe2x80x9cEnzymology of rat liver cytochromes P450xe2x80x9d, ed. Guengerich, F. P., CRC Press, Boca Raton Fla., Vol. 1, pp 1-54; Guengerich and Shimada (1991) Chem. Res. Toxicol. 4, 391-407; Gonzalez (1992) Trends in Pharmacol. Sci. 12, 346-352). The different mammalian P450s exhibit a unique but overlapping substrate specificity and display a high regio- and stereoselectivity (Crespi et al (1993) Toxicology 82, 89-104). Based on their major functions, mammalian P450s can be subdivided into two major classes: those involved primarily in the metabolism of steroids and bile acids, and those which mainly metabolise xenobiotics. The xenobiotic-metabolising P450s are typically located in the endoplasmic reticulum of certain mammalian cells such as liver cells and are termed microsomal P450s.
The compounds metabolised by the latter group of P450s include therapeutic drugs such as cyclosporin, nifedipine and debrisoquine as well as carcinogens such as polycyclic aromatic hydrocarbons, nitrosamines and arylamines. To be catalytically active, microsomal P450s require a supply of electrons which are shuttled from NADPH via the FMN and FAD prosthetic groups of NADPH-cytochrome P450 oxidoreductase (P450 reductase; EC 1.6.2.4) (Smith et al (1994) Proc. Natl. Acad. Sci. USA 91, 8710-8714).
Comparison of the primary structures of P450s indicates that they are structurally related to each other and are most likely derived from a common ancestor. Based on their primary structure the P450s are classified into families such as CYP1, CYP2 etc (Nelson et al (1996) Pharmacogenetics 6, 1-42).
Because of the importance of mammalian P450s in the metabolism of therapeutic compounds and carcinogens, attempts have been made to express mammalian P450s in heterologous systems. For example, mammalian cells have been used to express P450s heterologously as described by Doehmer et al (1988) Proc. Natl. Acad. Sci. USA 85, 5769-5773; Aoyama et al (1990) Proc. Natl. Acad. Sci. USA 87, 4790-4793; and Crespi et al (1991) Carcinogenesis 12, 355-359.
Yeast cells have also been used for the heterologous expression of cytochrome P450, for example by Renaud et al (1993) Toxicology 82, 39-52 and Bligh et al (1992) Gene 110, 33-39.
More recently mammalian P450s have been expressed in Escherichia coli. 
Gillam et al (1993) Arch. Biochem. Biophys. 305, 123-131 describes the expression of modified human cytochrome P450 3A4 in E. coli and purification and reconstitution of the enzyme.
Barnes et al (1991) Proc. Natl. Acad. Sci. USA 88, 5597-5601 describes the expression and enzymatic activity of recombinant cytochrome P450 17xcex1-hydroxylase in E. coli. 
Larson et al (1991) J. Biol. Chem. 266, 7321-7324 describes that expression of cytochrome P450 IIE1 lacking the hydrophobic NH2-terminal segment retains catalytic activity.
Shimada et al (1994) Carcinogenesis 15, 2523-2529 describes the activation of procarcinogens by human cytochrome P450 enzymes expressed in E. coli. 
Shet et al (1993) Proc. Natl. Acad. Sci. USA 90, 11748-11752 describes the enzymatic properties of a purified recombinant fusion protein containing NADPH-P450 reductase.
Shet et al (1995) Arch. Biochem. Biophys. 318, 314-321 describes some properties of a recombinant fusion protein containing the haem domain of human P450 3A4 and the flavin domains of rat cytochrome P450 reductase.
Jenkins and Waterman (1994) J. Biol. Chem. 269, 27401-27408 describes that flavodoxin and NADPH-flavodoxin reductase from E. coli support bovine cytochrome P450 c17 hydroxylase activities.
Fisher et al (1992) FASEB J. 6, 759-764 describes the expression of human cytochrome P450 1A2 in E. coli. 
Fisher et al (1992) Proc. Natl. Acad. Sci. USA 89, 10817-10821 describes the expression in E. coli of fusion proteins containing the domains of mammalian cytochromes P450 and NADPH-P450 reductase flavoprotein.
Chun and Chiang (1991) J. Biol. Chem. 266, 19186-19191 describes the expression of cholesterol 7xcex1-hydroxylase cytochrome P450 in E. coli. 
Richardson et al (1995) Arch. Biochem. Biophys. 323, 87-96 describes the expression of human and rabbit cytochrome P450s of the 2C subfamily in E. coli. 
Gillam et al (1995) Arch. Biochem. Biophys. 319, 540-550 describes the expression of cytochrome P450 2D6 in E. coli. 
Dong and Porter (1996) Arch. Biochem. Biophys. 327, 254-259 describes a study in which P450 reductase containing an N-terminal fusion to an ompA signal peptide is co-expressed in E. coli with human P450 2E1 in which the second codon of the P450 (serine) is replaced with an alanine; no other changes to the P450 were made. In vivo activity with whole cells could not be demonstrated.
WO 94/01568 describes the expression of P45017xcex1-hydroxylase in E. coli and also its fusion to a P450 reductase enzyme domain and expression of the fusion protein in E. coli. P450 enzyme hybrids, incorporating the N-terminal nine amino acids from bovine P45017xcex1-hydroxylase are also disclosed.
U.S. Pat. No. 5,240,831 describes expression of P45017xcex1-hydroxylase in E. coli in a biologically active form without the need for co-expression or admixture of a cytochrome P450 reductase.
Gillam et al (1995) Arch. Biochem. Biophys. 317, 374-384 describes the expression of cytochrome P450 3A5 in E. coli. 
Gillam et al (1994) Arch. Biochem. Biophys. 312, 59-66, describes the expression of modified human cytochrome P450 2E1 in E. coli. 
Shet et al (1994) Arch. Biochem. Biophys. 311, 402-417 describes a recombinant fusion protein expressed in E. coli containing the domains of bovine P450 17A and rat NADPH-P450 reductase.
Josephy et al (1995) Cancer Res. 55, 799-802 describes the bioactivation of aromatic amines by recombinant human cytochrome P450 1A2 expressed in Salmonella typhimurium. 
Despite the extensive efforts to express an effective eukaryotic, particularly mammalian, P450 monooxygenase enzyme system in bacteria to date no system which is capable of metabolising compounds in whole cells has been devised. This is particularly the case for xenobiotic-metabolising P450s which require a P450 reductase for enzymatic activity. More particularly, no bacterial cell system has previously been devised which allows cytochrome P450 and cytochrome P450 reductase to form a functional cytochrome P450 monooxygenase system when expressed separately in the same bacterial cell. Similarly, despite considerable efforts to produce bacteria which express a high level of a eukaryotic xenobiotic-metabolising P450, no satisfactory system has so far been devised.
One object of the invention is to provide superior systems for expressing P450s in a functional form in intact bacterial cells.
A further object of the invention is to provide an improved system for expressing P450s whether or not with P450 reductase.
Bacterial systems which express a functional P450 enzyme system in whole cells are useful as xe2x80x9cbioreactorsxe2x80x9d or they may find uses in drug-testing or carcinogen-testing systems or as biosensors or in environmental remediation or in the production of hormones and so on. The expression of eukaryotic P450s at high levels in bacteria provides a source of P450 for structural studies.
A first aspect of the invention provides a bacterial cell containing a functional cytochrome P450 monooxygenase system said cell comprising a genetic construct capable of expressing a cytochrome P450 and a genetic construct capable of expressing, separately from said cytochrome P450, a cytochrome P450 reductase wherein the N-terminus of the cytochrome P450 and the N-terminus of the cytochrome P450 reductase are each adapted to allow functional coupling of said cytochrome P450 and said cytochrome P450 reductase within said cell.
Preferably, the bacterial cell expresses a cytochrome P450 monooxygenase system which has a specific activity of at least 50 pmol/min/mg protein, more preferably at least 250 pmol/min/mg protein and still more preferably at least 500 pmol/min/mg protein. These levels are measured using a suitable and effective substrate for the cytochrome P450 monooxygenase system. Preferably the said preferred specific activities are of whole cells but the activities may also be those found in fractions of the cells such as the membrane fraction.
Thus, a bacterial cell is provided that contains a functional cytochrome P450 monooxygenase system said cell comprising a genetic construct which expresses a cytochrome P450 and a genetic construct which expresses, separately from said cytochrome P450, a cytochrome P450 reductase wherein the said cytochrome P450 and the said cytochrome P450 reductase functionally couple within said cell.
By xe2x80x9ccytochrome P450xe2x80x9d we include any haem-containing polypeptide which gives an absorption maximum in the region of 450 nmxc2x15 nm in a reduced CO difference spectrum by virtue of the formation of a CO adduct of the Fe(II) of said haem.
It is envisaged that the invention can be practised on any cytochrome P450.
Preferably, the cytochrome P450 is a eukaryotic cytochrome P450; more preferably the cytochrome P450 is a mammalian cytochrome P450 and still more preferably the cytochrome P450 is a human cytochrome P450.
A large number of cytochrome P450 cDNAs or genes have been cloned including a large number of eukaryotic cytochrome P450 cDNA, particularly mammalian cytochrome P450 cDNAs. For example, Nelson et al. (1996) Pharmacogenetics 6, 1-42, incorporated herein by reference, lists the known cytochrome P450 cDNA and genes and groups them into gene families and subfamilies based on the degree of sequence similarity and, to some extent, their chromosomal localization. Details of cytochrome P450 sequences are also available on the Internet.
These cytochrome P450 cDNAs and genes can readily be obtained using cloning methods well known in the art, some of which are described below and for example, described in Sambrook et al (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Press, Cold Spring House, New York.
It is particularly preferred if the cytochrome P450 is a member of any one of the cytochrome P450 CYP1, CYP2, CYP3 or CYP4 families. It is still further preferred if the cytochrome P450 monooxygenase system is one which metabolises xenobiotics.
At least some members of the cytochrome P450 families CYP1, CYP2 and CYP3 are involved in the metabolism of xenobiotic compounds such as therapeutic drugs. Members of the cytochrome P450 CYP1 family are involved in the metabolism of, for example, caffeine, benzphetamine, phenacetin, theophylline, acetaminophen, antipyrine, 2-hydroxyestradiol, imipramine, tamoxifen and Zoxazolamine. Members of the cytochrome CYP2 family are involved in the metabolism of, for example testosterone, aflatoxin, benzphetamine, cyclophosphamide, hexobarbital, 6-aminochrysene, retinol, tolbutamide(methyl), phenytoin, S-warfarin, tienilic acid, diazepam, propanalol, amitryptyline, bufuralol, bupranolol, clozapine, codeine, debrisoquine, desipramine, dextromorphan, ethylmorphine, flecainide, haloperidol, lidocaine, nortryptilline, propanolol, sparteine, taxol, tetrahydrocannabinol, progesterone and mephenytoin.
Members of the cytochrome CYP3 family are involved in the metabolism of, for example, lovastatin, nifedipine, taxol, teniposide, testosterone, verapamil, vinblastine, vincristine, vindesine, benzphetamine, cortisol, cyclosporin A and G, diazepam, dihydroergotamine, estradiol, ethynylestradiol, imipramine and lidocaine.
More preferably the cytochrome P450 is any one of the P450s CYP3A4, CYP2D6, CYP2A6, CYP2E1, CYP2D9 and CYP2C9.
Conveniently, the cytochrome P450, apart from the adaptation to its N-terminus, consists essentially of the same polypeptide sequence as the native cytochrome P450. However, the term xe2x80x9ccytochrome P450xe2x80x9d specifically includes modifications to native cytochrome P450, for example, modifications which alter the length or other properties of any hydrophobic N-terminal portion which may be present in the native cytochrome P450 or modifications, such as single or multiple point mutations or deletions, which modify the substrate specificity of the cytochrome P450 compared to the native cytochrome P450.
By xe2x80x9ccytochrome P450 reductasexe2x80x9d we include any NADPH-cytochrome P450 oxidoreductase which is able to transfer electrons from NADPH to cytochrome P450. Mammalian cytochrome P450 reductase contains one each of FMN and FAD prosthetic groups. It is preferred if the cytochrome P450 reductase is derived from the same species as the cytochrome P450 expressed in the bacterial cell. It is particularly preferred if the cytochrome P450 reductase is a mammalian cytochrome P450 reductase; rat or human cytochrome P450 reductase are especially preferred. The human cytochrome P450 reductase CDNA is described in Smith et al (1994) Proc. Natl. Acad. Sci. USA 91, 8710-8714. The rat cytochrome P450 reductase cDNA is described in Porter et al (1990) Biochemistry 29, 9814-9818.
Cytochrome P450 reductase cDNAs and genes can readily be obtained using cloning methods well known in the art, some of which are described below.
Conveniently, apart from the adaptation to its N-terminus, the cytochrome P450 reductase consists essentially of the same polypeptide sequence of the native cytochrome P450 reductase. However, the term xe2x80x9ccytochrome P450 reductasexe2x80x9d specifically includes modifications to native cytochrome P450, for example single or multiple point mutations or deletions which modify the cofactor binding.
It is preferred that when the cytochrome P450 is a human cytochrome P450 the cytochrome P450 reductase is human cytochrome P450 reductase.
By xe2x80x9cfunctional cytochrome P450 monooxygenase systemxe2x80x9d we mean that once the cytochrome P450 and cytochrome P450 reductase are expressed in the bacterial cell the cell, by virtue of the presence of said cytochrome P450 and said cytochrome P450 reductase, is able to convert a substrate for the cytochrome P450 monooxygenase system into a product provided that sufficient cofactors, such as NADPH and oxygen, are present.
By xe2x80x9cfunctional coupling of said cytochrome P450 and said cytochrome P450 reductase within the cellxe2x80x9d we mean that the cytochrome P450 and cytochrome P450 reductase are juxtaposed within the cell so that the cytochrome P450 reductase can provide electrons, whether directly or indirectly, to the cytochrome P450 during catalysis. The adaptation to the N-terminus of the cytochrome P450 or to the N-terminus of the cytochrome P450 reductase is merely one that allows functional expression and coupling of said cytochrome P450 and said cytochrome P450 reductase within the cell. The adaptation may be one which aids the juxtaposition of the cytochrome P450 and the cytochrome P450 reductase.
By xe2x80x9cwithin the cellxe2x80x9d we specifically include that the functional coupling may occur within or associated with any membrane or compartment of the cell including the periplasmic space or any membrane associated with the periplasm.
It is preferred if, the cytochrome P450 content of a culture of bacterial cells optimally expressing the cytochrome P450 is at least 100 nmol/l culture of whole cells, preferably at least 150 nmol/l culture of whole cells more preferably almost 250 nmol/l culture of whole cells, still more preferably about 500 nmol/l culture of whole cells and most preferably about 1000 nmol/l. Typically the cytochrome P450 content is around 200 nmol/l culture of whole cells.
Adaptions to each of the N-terminus of the cytochrome P450 and cytochrome P450 reductase which allow functional coupling of said cytochrome P450 and said cytochrome P450 reductase are discussed below.
Although not essential, it is preferred if the cytochrome P450 or cytochrome P450 reductase retains its own N-terminal sequence and that a further portion, such as a signal peptide as discussed below, is added to the N-terminus. of the cytochrome P450 or cytochrome P450 reductase or both.
Although it is envisaged that any aerobic or facultative anaerobic bacterial cell is suitable, it is preferred if the bacterial cell is Gram-negative and more preferred if the bacterial cell is a cell of a bacterium of the family Enterobacteriaceae, for example Escherichia coli. The Enterobacteria most closely related to E. coli are from the genus Salmonella and Shigella and less closely related are the genera Enterobacter, Serratia, Proteus and Erwinia. E. coli and S. typhimurium are the most preferred bacterial host cells for the present invention. Strains of E. coli K12 are most preferred because E. coli K12 is a standard laboratory strain which is non-pathogenic.
Although certain cytochrome P450 substrates are able to penetrate into the bacterial cell in order to be acted upon by the cytochrome P450 monooxygenase system it is preferred if the bacterial cell of the invention is one which has an increased permeability to a substrate or one which, when the bacterial cell is placed in an appropriate medium, becomes more permeable to the substrate. Additionally or alternatively it is preferred if the bacterial cell is one which has altered membrane properties, or a membrane whose properties can be altered, to facilitate the membrane penetration of the substrates. For example, a tolC mutant of E. coli has a more permeable membrane than a wild type E. coli (Chatterjee (1995) Proc. Natl. Acad. Sci. USA 92, 8950-8954) and the TA series of S. typhimurium strains have an increased permeability due to a deep rough mutation and have been frequently used for mutagenicity testing (see for example Simula et al (1993) Carcinogenesis 14, 1371-1376). S. typhimurium TA 97, 98, 100 and 102 as well as TA 1535 and TA 1538 have been used for mutagenicity testing in the pharmaceutical industry during drug safety evaluation. The present invention using these and other suitable bacterial strains are likely to improve these procedures, since they provide a humanized mutagenicity system and do not rely on rodent liver extracts (S9 fraction from rodent liver) as the metabolically activating system. The systems based on the present invention have also the advantage that the metabolically activating system and the target for mutagenicity, namely the DNA are within the same cell and are not physically separate entities as in the standard Ames test. This has the advantage that short lived metabolites are better detected and that the membrane barrier does not stop reactive metabolites to reach their DNA target. This, and other aspects of the invention, are discussed in further detail below.
It is also preferred if the cell is one which can be made more permeable by placing into the appropriate environment. Although there are many buffers systems which are suitable it is preferred if, following the expression phase, the bacterial cells, particularly E. coli cells, are resuspended in Tris-sucrose-EDTA, or TSE. TSE is 50 mM Tris. acetate (pH 7.6), 0.25 M sucrose, 0.25 mM EDTA. This may increase the permeability of the cells in several ways. Firstly, the cells are initially resuspended in double strength buffer, and then diluted rapidly with an equal volume of water. This has the effect of causing the release of periplasmic proteins, by rupturing the outer membrane momentarily. Secondly, EDTA is known to affect permeability directly, and can render cells more sensitive to certain hydrophobic agents. Thirdly, the Tris in the buffer can affect the structure of the lipopolysaccharide in the outer membrane, again altering permeability. Further details of ways to increase the permeability of the outer membrane of E. coli and Salmonella typhimurium is given in Nikaido and Vaara (1987) pp. 7-22 In: xe2x80x9cEscherichia coli and Salmonella typhimurium. Cellular and Molecular Biologyxe2x80x9d Vol. 1. Ed. Neidhardt, F. C., Am. Soc. Microbiol., Washington D.C.
Advantageously, especially when the bacterial cells are used in a bioreactor, the cells are solvent-resistant. Solvent-resistant E. coli cells are known in the art for example from Ferrante et al (1995) Proc. Natl. Acad. Sci. USA 92, 7617-7621.
It is preferred if the cytochrome P450 reductase comprises an N-terminal portion which directs the cytochrome P450 reductase to a cellular compartment or membrane of the bacterial cell. It is particularly preferred if the said N-terminal portion directs the cytochrome P450 reductase to a membrane.
It is preferred if the cytochrome P450 and the cytochrome P450 reductase are associated with a membrane in the bacterial cell. It is particularly preferred if the cytochrome P450 and the cytochrome P450 reductase are associated with the bacterial inner membrane (particularly in the case of E. coli and S. typhimurium), with their active sites located in the cytoplasm.
In one preferred embodiment the N-terminal portion is one which is derived from or based on an N-terminal portion of a bacterial protein wherein said bacterial protein is one which is directed to the periplasmic space or one which is destined for secretion from the bacterial cell. For example, the E. coli proteins encoded by the ompA, pelB, malE or phoA genes are such bacterial proteins. It is desirable if the presence of the N-terminal portion aids the correct folding of the cytochrome P450 or cytochrome P450 reductase.
Bacterial leader sequences or signal peptides which direct bacterial proteins usually into the periplasm have been fused previously to the N-terminus of a few mammalian proteins with a view to exporting the resulting fusion proteins to the oxidising environment of the periplasm. Thus, such bacterial leader sequences or signal peptides have been used in the expression of mammalian secretory proteins such as immunoglobulins or fragments thereof. In contrast, mammalian xenobiotic-metabolising cytochrome P450s, when found in the endoplasmic reticulum in nature, are usually exposed to a reducing environment.
Thus, a particularly preferred embodiment is wherein the N-terminal portion comprises any one of the ompA, pelB, malE or phoA signal peptides or leader sequences or a functionally equivalent variant thereof.
By xe2x80x9cfunctionally equivalent variant thereofxe2x80x9d we include any peptide sequence which, if present in place in the native said bacterial protein, would direct said protein to the same cellular location as the natural signal peptide.
It is preferred if the N-terminal portion of each of the cytochrome P450 and the cytochrome P450 reductase is a signal peptide or signal-peptide like N-terminal portion.
It is particularly preferred if the N-terminal portion is one which competes with the ompA leader for the general secretory pathway or competes with ompA for the signal recognition machinery, including the signal recognition particle and trigger factor.
The general secretory pathway and components thereof are described in Pugsley (1993) Microbiol. Rev 57, 50-108, incorporated herein by reference, and the signal recognition particle and trigger factor are described in Valent et al (1995) EMBO J. 14, 5494-5505, incorporated herein by reference. Competition assays between ompA signal peptide and a putative signal peptide can be carried out using methods known in the art using the teaching of Pugsley and Valent et al.
The pelB leader sequence consists of the amino acid sequence MKYLLPTAAAGLLLLAAQPAMA (SEQ ID No 1).
The ompA leader sequence consists of the amino acid sequence MKKTAIAIAVALAGFATVAQA (SEQ ID No 2).
Signal peptides have certain recognisable common features, which are detailed in von Heijne (1986) Nucl Acids Res 14, 4683-4690; Gierasch (1989) Biochemistry 28, 923-930; and the chapter by Oliver (1987) on Periplasm and Protein Secretion, pp. 56-69. Oliver (1987) In: xe2x80x9cEscherichia coli and Salmonella typhimurium Cellular and Molecular Biologyxe2x80x9d, Vol. 1, Ed. Neidhardt, F. C., Am. Soc. Microbiol, Washington D.C. Firstly, there is an N-terminal net positively charged region of variable length (n-region). This is followed by a hydrophobic core (h-region), of 10xc2x13 amino acids, which is rich in Leu, Ala, Met, Val, Ile, Phe and Trp residues. Finally, there is the c-region of typically 5-7 amino-acids, which are generally slightly more polar than those in the h-region. The most important amino acids in this c-region are those at the xe2x88x923 and xe2x88x921 positions relative to the site of signal cleavage (the xe2x80x9cxe2x88x923, xe2x88x921 rulexe2x80x9d) xe2x80x94there appear to be severe constraints on the possible amino-acids which can exist in these positions: only those with small side-chains are tolerated. Thus, only Ala, Gly, Leu, Ser, Thr and Val have been found at xe2x88x923 (with Ala strongly preferred), and only Ala, Gly, Ser and Thr at xe2x88x921 (with Ala again strongly preferred). Evidence suggests that xcex2-turn formation in this signal processing region is important for signal cleavage to occur (see, for example, Barkocy-Gallagher et al (1994) J Biol Chem 269, 13609-13613, and Duffaud and Inouye (1988) J Biol Chem 263, 10224-10228.
It is preferred if signal peptide cleavage occurs. Thus, it is preferred if a suitable amino-acid sequence immediately downstream of the signal peptide, ie. in the protein to be expressed is included, since this still forms part of the xe2x80x9ccleavage sitexe2x80x9d. For example, a proline in position +1 inhibits signal removal (Barkocy-Gallagher et al (1992) J. Biol. Chem. 267, 1231-1238). Using ompA as an example, it may be that to ensure complete cleavage, if this is desirable, that the first few amino-acids of the mature ompA protein in the construct, immediately after the ompA leader, and immediately before the P450 or reductase sequence is included. Signal peptide cleavage is caused by a specific signal peptidase enzyme, for example signal peptidase I. In a preferred embodiment signal peptidase I is overproduced in the bacterial cell (for example, E. coli) to aid signal peptide cleavage if this is desirable (see van Dijl et al (1991) Mol. Gen. Genet. 227, 40-48).
It is particularly preferred if the first two amino acids of the mature OmpA protein (ie. Ala Pro) are inserted immediately downstream of the ompA signal peptide and before the N-terminus of the P450.
Other preferred possibilities of increasing the probability of signal peptide cleavage is to introduce a short linker sequence between the signal peptide and the P450, or expression in different strains may lead to increased or reduced signal peptide cleavage compared with, for example, expression in DH5xcex1.
Thus it will be seen that it is preferred if the N-terminal portion is a signal peptide which when present in its natural polypeptide has the function to mediate the membrane insertion and the export of the national polypeptide through the cytoplasmic membrane into the periplasmic space. Sequence of the leader sequence or signal peptide is usually up to 40 amino acid residues. It is envisaged that leaders can be modified without altering their functional properties.
In a further embodiment of the invention it is also preferred if the cytochrome P450 comprises an N-terminal portion which directs the cytochrome P450 to a cellular compartment or membrane of the bacterial cell. It is particularly preferred if the said N-terminal portion directs the cytochrome P450 to a membrane.
The preferred N-terminal portions in this embodiment are the same as the preferred N-terminal portions for the cytochrome P450 reductase. It is particularly preferred if the N-terminal portion of the cytochrome P450 comprises any one of the ompA, pelB, malE or phoA signal peptides or leader sequences, or a functionally equivalent variant thereof. The ompA signal peptide is particularly preferred.
In a further particularly preferred embodiment the N-terminal portion of the cytochrome P450 and the N-terminal portion of the cytochrome P450 reductase each direct the said cytochrome P450 or cytochrome P450 reductase to the same cellular compartment or membrane. This is particularly advantageous because it increases or improves the functional coupling of said cytochrome P450 and said cytochrome P450 reductase within the cell. It is particularly preferred if the cytochrome P450 and cytochrome P450 reductase are directed to the cytoplasmic side of the inner membranes where access is gained to the bacterial cytoplasmic NADPH pool.
In further preference the N-terminal portion of the cytochrome P450 is substantially the same as the N-terminal portion of the cytochrome P450 reductase.
In a further embodiment the cytochrome P450 comprises an N-terminal portion which is adapted to increase the translatability or correct folding of said cytochrome P450 in said bacterial cell.
Preferably, the cytochrome P450, compared to its native sequence, is modified at the N-terminus thereof.
By xe2x80x9ctranslatabilityxe2x80x9d we mean the efficiency with which a given RNA molecule can be translated into a polypeptide.
There are several features of the optimised CYP3A4 sequence which improve translatability.
These features include:
1. Second codon changed to suit E. coli preference (often GCT, encoding Ala). This is demonstrated by Looman et al (1987) EMBO J 6, 2489-2492.
2. Codons 4 and 5 made rich in A and T residues (where possible), to minimise the potential for mRNA secondary structure around the start codon. Minimisation of mRNA structure around the ribosome binding site and start codon can have large effects on the xe2x80x9ctranslatabilityxe2x80x9d (see, for example, Wang et al (1995) Protein Expr Purif 6, 284-290.
Concerning the use of leader sequences, the principal advantage is that by their nature, they are already xe2x80x9coptimisedxe2x80x9d for bacterial expression, since they come from bacterial genes. This often includes the two features described above for example, both pelB and ompA leaders contain AAA (Lys) as the second codon, which was the best performing second codon in the paper of Looman et al in terms of xe2x80x9ctranslatabilityxe2x80x9d. In addition, they often have reduced secondary structure around the ribosome binding site and start codon (see, for example, Movva et al (1980) J Mol Biol 143, 317-328 on ompA gene structure).
An additional advantage of using an N-terminal signal peptide fusion concerns the minimisation of any effect of rare codons in the P450 or reductase cDNA. For example, the AGA/AGG codon is the least used in E. coli (Chen and Inouye (1990) Nucl Acids Res 18, 1465-1473), and slows down translation as a result of the limiting availability of the corresponding charged tRNA molecule. However, the negative effect of such a rare codon reduces as its distance from the start codon increases (Chen and Inouye, supra). By adding an xe2x80x9coptimisedxe2x80x9d bacterial leader sequence (typically 20-25 amino-acids in length) to the N-terminus of the P450 (or reductase), any rare codon close to the 5xe2x80x2-end of the P450 cDNA will be moved that much further away from the start codon, and therefore have much less effect.
Thus, it is preferred if xe2x80x9ctranslatabilityxe2x80x9d is improved by one or more of the following means:
1. Removal of rare codons (see Chen and Inouye, supra, such as AGG/AGA (Arg), CUA (Leu), AUA (Ile), CCC (Pro), and GGA/GGG (Gly) from the P450 or reductase cDNA, especially those less than 25-30 codons from the start codon.
2. As an alternative to, or in conjunction with, the above, introduction of genes encoding the rare tRNA synthases, for example, the dnaY gene for AGG/AGA.
3. Making other changes to the DNA sequence to reflect E. coli preferences, for example, the non-random utilisation of codon pairs (Gutman and Hatfield (1989) Proc Natl Acad Sci USA 86, 3699-3703).
4. Making changes to the promoter/ribosome binding site within the expression vector in order to minimise secondary structure potential and to optimise the distance between the ribosome binding site and the start codon (see Wang et al (1995), supra).
It is also preferred if the cytochrome P450 has its N-terminus modified according to U.S. Pat. No. 5,240,831 or, for example, by the general method of Gillam et al (1995) Arch. Biochem. Biophys. 317, 374-384, both of which are incorporated herein by reference.
The bacterial cell of the invention comprises a genetic construct capable of expressing a cytochrome P450 and a genetic construct capable of expressing a cytochrome P450 reductase.
Conveniently, the cell may contain one genetic construct capable of expressing both a cytochrome P450 and a cytochrome P450 reductase or the cytochrome P450 and the cytochrome P450 reductase may be expressed from separate genetic constructs.
The genetic construct may be DNA or RNA. DNA is preferred.
The genetic construct is typically an extrachromosomal genetic element such as a plasmid or bacteriophage genome but the term xe2x80x9cgenetic constructxe2x80x9d specifically includes that the genetic construct may be part of the bacterial chromosome. For example, the genetic construct may be part of a bacteriophage which has lysogenised the bacterial chromosome.
The genetic construct comprises those genetic elements which are necessary for expression of the cytochrome P450 or cytochrome P450 reductase in the bacterial cell.
The elements required for transcription and translation in the bacterial cell include a promoter, a ribosome binding site, a coding region for the cytochrome P450 or cytochrome P450 reductase.
In terms of the promoter, it is believed that virtually any promoter functional in the selected bacterial cell may be employed. However, preferred promoters include the lac, lac UV5, tac, trc, xcexPL, T7, lpp, lpp-lac or T3 promoter. Of course, the xcexPL, T7 and T3 promoters are derived from bacteriophage and are known to be functional in bacteria such as E. coli. 
The use of a regulatable promoter, such as the lac promoter, is preferred. It is preferred if a strong promoter, such as the T7 promoter, is not used.
One will often desire to incorporate an appropriate ribosome binding site for effecting bacterial expression into the eukaryotic cytochrome P450 domain comprising gene. Often, the ribosome binding site and promoter can be incorporated as a xe2x80x9ccassettexe2x80x9d, defined as a contiguous, pre-fabricated DNA segment which incorporates the desired elements and has useful restriction enzyme recognition sites at its two termini, allowing it to be readily inserted at an appropriate point within the desired cytochrome P450 gene or cDNA or cytochrome P450 reductase by simple genetic manipulation.
Most conveniently, one may desire to simply employ a promoter and ribosome binding site from a homologous system, such as the lac promoter and its associated RBS. In general, however, it is proposed that one may employ any effective bacterial ribosome binding site, with those RBSs from E. coli, xcex, T7 or T3 being preferred. Even more preferred ribosome binding sites are those from the T7 gene 10, or E. coli lac A, lac Z, trp A, trp B, trp C, trp D, trp E, trp L, trp R or trp S genes. A particularly preferred ribosome binding site and spacer region comprises the sequence 5xe2x80x2-AGGAGGTCAT-3xe2x80x2 (SEQ ID No 3), wherein the underlined portion comprises the ribosome binding site and the adjacent CAT sequence comprises the spacer region. (The spacer region is that sequence between the ribosome site and the ATG initiation codon.)
One will also typically desire to incorporate an appropriate bacterial transcription terminator, which functions to terminate the function of bacterial RNA polymerases, the enzymes responsible for transcribing DNA into RNA into a gene prepared in accordance with the invention. The requirements for a functional bacterial transcription terminator are rather simple, and are usually characterized by a run of T residues preceded by a GC rich dyad synunetrical region. The more preferred terminators are those from the TRP gene, the ribosomal terminators, rrnB, or terminator sequences from the T7 phage. In fact, the T7 terminator sequences contain RNase III cleavage sites with a stem-loop structure at the 3xe2x80x2 ends of mRNAs which apparently slows down message degradation.
The genetic construct is capable of propagation in the bacterial cell and is stably transmitted to future generations.
A variety of methods have been developed to operably link DNA to vectors via complementary cohesive termini For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.
Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors. The DNA segment, generated by endonuclease restriction digestion as described earlier, is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3xe2x80x2-single-stranded termini with their 3xe2x80x2-5xe2x80x2-exonucleolytic activities, and fill in recessed 3xe2x80x2-ends with their polymerizing activities.
The combination of these activities therefore generates blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the products of the reaction are DNA segments carrying polymeric linker sequences at their ends. These DNA segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment.
Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, Conn., USA.
A desirable way to modify the DNA encoding the polypeptide of the invention is to use the polymerase chain reaction as disclosed by Saiki et al (1988) Science 239, 487-491.
In this method the DNA to be enzymatically amplified is flanked by two specific oligonucleotide primers which themselves become incorporated into the amplified DNA. The said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.
Suitably, the vectors include a prokaryotic replicon, such as the ColE1 ori, for propagation in a prokaryote. The vectors can also include an appropriate promoter such as a prokaryotic promoter capable of directing the expression (transcription and translation) of the genes in a bacterial host cell, such as E. coli, transformed therewith.
A promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with exemplary bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.
Typical prokaryotic vector plasmids are pUC18, pUC19, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, Calif., USA) and pTrc99A and pKK223-3 available from Pharmacia, Piscataway, N.J., USA.
Transformation of appropriate cell hosts with a DNA construct of the present invention is accomplished by well known methods that typically depend on the type of vector used. With regard to transformation of bacterial host cells, see, for example, Cohen et al (1972) Proc. Natl. Acad. Sci. USA 69, 2110 and Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Electroporation is also useful for transforming cells and is well known in the art for transforming bacterial cells.
For example, many bacterial species may be transformed by the methods described in Luchansky et al (1988) Mol. Microbiol. 2, 637-646 incorporated herein by reference. The greatest number of transformants is consistently recovered following electroporation of the DNA-cell mixture suspended in 2.5xc3x97 PEB using 6250 V per cm at 25 xcexcFD.
Successfully transformed cells, ie cells that contain a DNA construct capable of expressing said cytochrome P450 or cytochrome P450 reductase, can be identified by well known techniques. For example, cells resulting from the introduction of an expression construct of the present invention can be grown to produce the cytochrome P450 or cytochrome P450 reductase. Cells can be harvested and lysed and their DNA content examined for the presence of the DNA using a method such as that described by Southern (1975) J. Mol. Biol. 98, 503 or Berent et al (1985) Biotech. 3, 208. Alternatively, the presence of the protein in the supernatant can be detected using antibodies as described below.
In addition to directly assaying for the presence of recombinant DNA, successful transformation can be confirmed by well known immunological methods when the recombinant DNA is capable of directing the expression of the protein. For example, cells successfully transformed with an expression vector produce proteins displaying appropriate antigenicity. Samples of cells suspected of being transformed are harvested and assayed for the protein using suitable antibodies.
Thus, in addition to the transformed host cells themselves, the present invention also contemplates a culture of those cells, preferably a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium.
In a further preferred embodiment the bacterial cell further comprises a genetic construct capable of expressing a polypeptide cofactor which aids the correct folding of the cytochrome P450 or the cytochrome P450 reductase.
The presence of such a polypeptide cofactor is especially preferred when expressing the cytochrome P450 or the cytochrome P450 reductase from a strong promoter where, in the absence of the polypeptide cofactor, the cytochrome P450 or cytochrome P450 reductase may form non-functional inclusion bodies.
Preferably, the polypeptide cofactor is a molecular chaperone such as the Gro ELS complex, SecB, SecD, SecF, the DnaJ/DnaK/GrpE complex, peptidylprolyl-cis, trans isomerases, protein disulphide isomerase-like proteins encoded by the genes dsbA, dsbB, dsbC and dsbD, the periplasmic chaperone encoded by clpB or thioredoxin. The solubility of foreign proteins expressed in E. coli has been increased by the coproduction of bacterial thioredoxin (Yasukawa et al (1995) J. Biol. Chem. 270, 25328-25331; incorporated herein by reference).
It may also be useful to use the following systems or host or expression systems.
1. The use of protease-deficient strains of E. coli (for example, ompTxe2x88x92, lonxe2x88x92, degPxe2x88x92), which may reduce degradation of the expressed protein(s). A set of E. coli strains deficient in all known loci affecting proteolytic of secreted recombinant proteins is described in Meerman and Georgion (1994) Biotechnology 12, 1107-1110.
2. The expression of the P450 and/or reductase as a fusion protein with, for example, ubiquitin (Baker et al (1994) J. Biol. Chem. 269, 25381-25386), thioredoxin (eg pTrxFus vector from Invitrogen), glutathione S-transferase (eg pGEX vectors from Pharmacia) or protein A (eg pRIT2T vector from Pharmacia), any of which may enhance expression.
In a still further preferred embodiment the bacterial cell further comprises a genetic construct capable of expressing a polypeptide cofactor which aids transfer of electrons between the cytochrome P450 and the cytochrome P450 reductase. Preferably, the said cofactor is cytochrome b5 or the FMN domain of a cytochrome P450 reductase.
Other cofactors which may aid the transfer of electrons include adrenodoxiniadrenodoxin reductase and NADH cytochrome b5 reductase (in conjunction with cytochrome b5). It is believed to be of particular benefit to use a cofactor which takes electrons from NADH, rather than NADPH in the present invention, or even to use both cofactors together, especially when dealing with whole cell metabolism (xe2x80x9cbioreactorsxe2x80x9d). This is because the intracellular ratio of (NAD+NADH) to (NADP+NADPH) in E. coli is about 4:1. Therefore, there is a far greater potential for P450 reduction (and therefore enzyme activity) if the reducing equivalent is NADH rather than NADPH. In conjunction with this, the invention includes additions or modifications which may lead to an increase in the cytoplasmic pool of NADH and/or NADPH as a means of increasing enzyme activity in whole cells. This includes addition of precursors to the extracellular medium (those for which an uptake mechanism exists, such as nicotinamide), or inhibition of enzymes which destroy NADPH.
The FMN domain of cytochrome P450 reductase can be expressed as described in Smith et al (1994) Proc. Natl. Acad. Sci. USA 91, 8710-8714 and cytochrome b5 can be expressed as described in Holmans et al (1994) Arch. Biochem. Biophys. 312, 554-565. It is preferred if a polypeptide cofactor which aids transfer of electrons is included when the cytochrome P450 is any one of CYP3A4, CYP3A5, CYP3A7, CYP2E1 and CYP1A1. It is particularly preferred if cytochrome b5 is co-expressed with any one of CYP3A4, CYP3A5, CYP3A7 or CYP2E1.
It is also particularly preferred if the FMN domain is co-expressed with CYP1A1.
Although it is envisaged that in some instances the said cofactor may comprise an N-terminal portion which directs the cofactor to a cellular compartment or membrane of the bacterial cell, it is preferred if no such modifications are made to cytochrome b5 or the FMN domain of cytochrome P450 reductase when they are expressed in the bacterial cell.
A further embodiment comprises a bacterial cell of the invention further comprising a genetic construct capable of expressing any one of an enzyme capable of metabolising the product of a reaction catalysed by the cytochrome P450 monooxygenase system.
In order to attempt to mimic to metabolism of a compound by a eukaryotic, especially mammalian, cell or an organ from an animal, especially a mammal, it is desirable to express in the bacterial cell of the invention one or more further polypeptides which, in the eukaryotic cell or in the animal may metabolise further the product of the cytochrome P450 monooxygenase system. This is particularly beneficial when the bacterial cell of the invention is used for mutagenicity testing or as a model for drug metabolism.
Conveniently, the enzyme is any of a glutathione S-transferase, an epoxide hydrolase or a UDP-glucuronosyl transferase. Other enzymes include sulfotransferase, N-acetyltransferase, alcohol dehydrogenase, xcex3-glutamyl transpeptidase, cysteine conjugate xcex2-lyase, methyltransferase, thioltransferase, DT-diaphorase, quinone reductase or glyoxalase.
It will be appreciated that because several genetic constructs may be present in the same bacterial cell, for example constructs expressing cytochrome P450 and cytochrome P450 reductase and sometimes also cytochrome b5 or an FMN domain of cytochrome P450 reductase or a further enzyme, it is convenient if bacterial strains are provided which have one or more of the genetic constructs integrated into their chromosome and that these strains can then be used as a xe2x80x9cmasterxe2x80x9d strain for the introduction of further genetic elements. For example, it is particularly preferred if the bacterial xe2x80x9cmasterxe2x80x9d strains comprise a cytochrome P450 reductase genetic construct integrated into the bacterial chromosome. It is also preferred if the bacterial xe2x80x9cmasterxe2x80x9d strains comprise a genetic construct or constructs which express both cytochrome P450 reductase and cytochrome b5 from the bacterial chromosome. These xe2x80x9cmasterxe2x80x9d strains are then transformed with a genetic construct capable of expressing a cytochrome P450.
A further aspect of the invention provides a method of culturing a cell of the first aspect of the invention. Any suitable culture medium may be used. It is preferred if a nutrient-rich broth, such as Terrific broth, is used. It is also preferred if the culture medium contains a compound which aids haem synthesis; xcex4-amino levulinic acid (ALA) is particularly preferred.
It will be appreciated that in all aspects of the invention where a bacterial cell contains two or more genetic constructs those genetic constructs are compatible with each other in the same bacterial cell. In general, genetic constructs which are integrated into the chromosome are compatible with one another and two genetic constructs are usually compatible with one another when one is integrated into the chromosome and the other is an autonomous replicon such as a plasmid. In general, when there are two or more different plasmids without the same cell which constitute the genetic constructs of the invention it is desirable if they are compatible plasmids, for example plasmids which have different origins of replication. It is also desirable if the different plasmids encode different antibiotic resistance genes so that all of the different plasmids can be selected when the bacterial cell is grown in culture.
A second aspect of the invention provides a method of converting a substrate for cytochrome P450 into a product, the method comprising admixing said substrate with a bacterial cell according to the first aspect of the invention wherein said cell contains a functional cytochrome P450 monooxygenase system which can convert said substrate.
A third aspect of the invention provides the use of a bacterial cell according to the first aspect of the invention for converting a substrate of a cytochrome P450 into a product.
The bacterial cells of the invention will find uses in many fields of technology, particularly those cells of the first aspect of the invention which express a functional cytochrome P450 monooxygenase system.
The following are some specific uses to which the bacterial cells of the invention can be put but it is envisaged that there are many other uses, for example whenever it is desirable to convert a cytochrome P450 substrate into a product.
a) Drug Development and Drug Testing
Bringing safe new drugs onto the market is expensive, complicated and protracted. Efficacy and safety of the market product, with respect to pharmacokinetic parameters, drug/drug interactions and toxicity, are critically dependent on the models employed for drug development. A major advance in drug development would result if the shortcomings of lead compounds could be predicted at the earliest stage of development.
There are serious problems in extrapolating pharnacotoxicological data from animal models to man. These are often due to pronounced species differences in the catalytic properties of drug metabolizing enzymes, which determine the pharmacological and the toxicological properties of most therapeutic drugs.
The bacterial cells, particularly E. coli and S. typhimurium cells, which form part of the present invention and which express functional P450 monooxygenase systems, are ideal models for mimicking human drug metabolism and are easier to handle than yeast and mammalian cell based models. These cells allow the high throughput screening of drugs with respect to optimized drug metabolism properties. This issue becomes particularly important with the advent of combinatorial chemical libraries which necessitate the evaluation of the drug metabolism properties of several hundred compounds within a short time.
In this embodiment it is useful if the bacterial cells also express other drug-metabolizing enzymes as described herein.
b) Bioreactors
The bacterial cells of the invention, because of the high substrate, region and stereoselectivity of the oxidative reactions catalyzed by P450s are useful for the synthesis of fine or bulk chemicals and the synthesis of intermediates of chemical reactions.
In this embodiment it is clear that a bacterial cell of the invention is selected which expresses a cytochrome P450 with the appropriate substrate specificity. The substrate specificity of many cytochrome P450s is known in the art and so the appropriate cytochrome P450 can be readily selected. However, as more cytochrome P450 genes are found it will be possible to use those in the invention and, indeed, the bacterial cells of the invention, which express a new cytochrome P450 can be used to determine its substrate specificity.
It will be appreciated that because some cytochrome P450s are able to convert alkanes to alcohols or aromatic compounds to phenolic compounds the bacterial cells of the invention are useful in the bulk chemical industry where such alcohols and phenolic compounds are required. However, many of the reactions catalysed by cytochrome P450s make the cells of the invention useful in the fine chemical and pharmaceutical industries where selective oxidation (including hydroxylation) of complex structures is often required. It is believed that the cells of the invention are particularly suited to the synthesis of steroid hormones and analogues thereof.
c) Biocatalysis
The systems developed in the present invention will allow rapid functional testing of P450 variants generated by site directed mutagenesis. It will be therefore possible to generate within a short time novel P450s with improved catalytic properties.
d) Bio- and Chemo-sensors
The bacterial cells of the invention are also useful as bio- or chemo-sensors. In particular membranes isolated from the cells are useful. Binding of substrates (which are the molecules to be sensed or detected) can cause a change of potential when the bacterial cell or the membranes isolated from the cell are present on an electrode surface thereby allowing detection of the substrate molecule. The use of immobilised cells for detection and analysis is described in Kambe and Nakanishi (1994) Current Opinion in Biotechnology 5, 54-59.
e) Bioremediation
The bacterial cells of the invention are also useful in bioremediation. For example, cytochrome P450 monooxygenase systems are able to detoxify harmful compounds. The appropriate bacterial cells expressing an appropriate cytochrome P450 which can oxidise the harmful compound is useful in rendering the said compound less harmless.
f) Carcinogenicity Testing
As is described in more detail else where, the cells of the invention, particularly S. typhimurium cells, are useful in carcinogenicity testing.
Although it is envisaged that the cells of the invention are especially useful because they provide a functional cytochrome P450 monooxygenase system within an intact cell, it is also part of the invention that membranes are isolated from said cells and that said membranes are enriched in the cytochrome P450 monooxygenase system compared with whole cells. Membrane isolation from bacterial cells is well known in the art. Membrane isolation from cells of the invention is described in more detail in the Examples.
A fourth aspect of the invention provides a bacterial cell containing a cytochrome P450 said cell comprising a genetic construct capable of expressing, said cytochrome P450 wherein the cytochrome P450 comprises an N-terminal portion which directs the cytochrome P450 to a cellular compartment or membrane of the bacterial cell.
It is preferred if the N-terminal portion directs the cytochrome P450 to a membrane.
It is further preferred if the N-terminal portion comprises the ompA, pelB, malE or phoA signal peptide or a functionally equivalent variant thereof.
The preferred features of the N-terminal portion, particularly those of the signal peptides or leader sequences, are those preferred in the previous aspects of the invention.
It is still further preferred if the cytochrome P450 further comprises a peptide sequence which will aid purification of the cytochrome P450 from the bacterial cell; more preferably wherein said peptide sequence comprises a binding site for a compound.
It is particularly preferred if said peptide sequence is a -(His-)n where n xe2x89xa74 and said compound is nickel.
It is contemplated that the fourth aspect of the invention is useful both for those cytochrome P450s which ordinarily couple with cytochrome P450 reductase and for other cytochrome P450s such as those which couple adrenodoxin/adrenodoxin reductase or equivalent electron transfer compounds. Thus, in a preferred embodiment of the fourth aspect of the invention, the cell further comprises a genetic construct capable of expressing each, or both, of adrenodoxin or adrenodoxin reductase of equivalent electron transfer components. By xe2x80x9cequivalent electron transfer componentsxe2x80x9d we include all other functionally-equivalent components which can transfer electrons from NADH to cytochrome P450, particularly those components whose natural function is to transfer electrons from NADH to particular cytochrome P450s.
A fifth aspect of the present invention provides a method of preparing cytochrome P450, the method comprising the steps of (a) providing a sufficient quantity of cells according to the fourth aspect of the invention and (b) separating the cytochrome P450 from the other cellular compartments.
A further aspect of the invention provides a genetic construct capable of expressing a cytochrome P450 wherein the cytochrome P450 comprises an N-terminal portion which directs the cytochrome P450 to a cellular compartment or membrane of a bacterial cell. The preferred features of the N-terminal portion are those preferred in relation to the other aspects of the invention. It is particularly preferred if the N-terminal portion comprises the ompA, pelB, melE or phoA signal peptide or a functionally equivalent variant thereof. Other preferred features of the genetic construct are those preferred in the previous aspects of the invention. A still further aspect of the invention provides a plurality of bacterial cells of the first or fourth aspects of the invention, each cell containing a genetic construct capable of expressing a different cytochrome P450 or containing a genetic construct or constructs which encode different combinations of cytochrome P450 and cytochrome P450 reductase and, if appropriate, other polypeptides such as those which aid electron transfer or those which will further metabolise the product of the reaction of the cytochrome P450 monooxygenase system with a substrate.
Such a plurality (or library) of cells can conveniently be stored, for example in suitable conditions in a freezer and, for example, in a microtitre plate, each well containing a different bacterial cell. The plurality of cells may be useful for drug-testing or carcinogenicity testing and for other purposes such as those described above.